Clinical and basic laboratory studies are directed at developing efficient and safe gene transduction and ex vivo manipulation strategies for hematopoietic cells, including stem and progenitor cells and lymphocytes. During the past year we completed an ongoing clinical trial of retroviral gene transfer into CD34+ cells from patients with breast cancer, comparing survival of cells containing a drug-resistance gene to those receiving a control gene. Cells containing the drug resistance gene survived preferentially after chemotherapy. In the rhesus model, shown to be the only predictive assay for human clinical results, we have focused on optimizing gene transfer to primitive stem and progenitor cells, and using genetic marking techniques to understand stem cell behavior in vivo. We have been encouraged to discover that the inclusion of the cytokine flt 3 ligand and either autologous stromal cells or fibronectin improves gene transfer efficiency into engrafting cells to levels of 10-50%, a range with clinical utility. These high levels have also allowed for the first time in a large animal model assessment of contribution of specific marked clones to different lineages over time, using inverse PCR and insertion site analysis. Over 40 clones contribute to multiple lineages for a year thus far, which has clinical implications and is a surprise based on rodent data. Other ongoing projects in the lab involve the assessment of the effect of foreign expressed genes in vector constructs on levels of gene-modified cells in vivo, using either CD34+ primitive cells or lymphocytes as targets. We have shown that tolerance to foreign gene products can be induced by introducing the gene into stem cells, as opposed to mature lymphocytes. - STEM CELLS, GENE THERAPY, RETROVIRUSES, CYTOKINES